Coupling agents are useful in the area of diagnosis, in particular to detect reactions of ligand-anti-ligand type such as antigen-antibody or protein-protein, since they allow the direct coupling of a molecule of biological interest, such as an antigen, with a detecting molecule such as an enzyme. The binding of the molecule of biological interest to another molecule such as an antibody is evidenced by means of the detecting molecule.
For this purpose, coupling agents must be bifunctional having both a chemical function enabling their coupling with said molecule of biological interest and a further chemical function enabling their coupling with said detecting molecule, and must also have a spacer arm allowing sufficient distancing of the coupled molecules.
Various heterobifunctional coupling agents are known from the prior art. These coupling agents, found in the PIERCE catalogue for example, essentially have a maleimide function, a hydrazone function and a spacer arm of saturated or aromatic alkyl type. These coupling agents are used for coupling molecules having free thiol functions and also for coupling molecules having aldehyde or ketone functions. However, these coupling agents have the disadvantage of being scarcely soluble in aqueous media in particular on account of the hydrophobic nature of the spacer arm, and often require the use of organic co-solvents when used, in particular to prepare conjugates. In addition, the proteins coupled to these coupling agents have a tendency to precipitate during marking.
Mikolajczyk S. et al. (Bioconjugate Chemical, 1994, 5(6), 636–646) describe the use of a coupling agent having a maleimide function, an aminooxyalkylene function and a relatively short spacer arm containing L-lysine in particular. This coupling agent is used for coupling two modified proteins, β-lactamase and the Fab murine peptide fragment. The disadvantages of this coupling agent are that it has a short spacer arm not allowing sufficient distancing of the molecules coupled to the coupling agent, and its synthesis method is long. Also a stability problem of the coupling agent may arise on account of the presence of the lysine —COOH function which could react with the alcoxyamine function known to be highly reactive.